Measurement of k(L)a by dynamic pressure method in pilot-plant fermentor

The dynamic pressure method (DPM) is used for measurement of k(L)a in a 1-m(3) pilot scale fermentor in coalescing (distilled water) and noncoalescing (0.3 M Na(2)SO(4) aqueous solution) batches. The method consists in recording oxygen concentration in a batch after a small pressure change (20 kPa) in the fermentor. The upward pressure change is brought about by temporary closing and subsequent throttling of outlet gas stream and the downward change by full reopening of the gas outlet. Absorption of pure oxygen yields the same k(L)a values as absorption of air. In noncoalescing batch, the downward k(L)a values are always higher than the upward values owing to spontaneous nucleation of bubbles. The experiments performed in a stirred cell confirm this behavior. Thus, only upward pressure change should be used for measurement. The correlation of k(L)a data measured in small (18-L) and large (1000-L) vessels based on power dissipated and superficial gas velocity are in a good agreement. Unlike the DPM, the classical dynamic methods yield, under the same conditions, excessively low values of k(L)a (the dynamic startup method) or fail to produce data at all (the dynamic method with interchange of air for N(2)). (c) 1994 John Wiley & Sons, Inc.     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Symmetric enzyme distribution in asymmetric UF polysulfone membranes

Invertase as well as as amyloglucosidase were immobilized within asymmetyric ultrafiltration membranes that were prepared from polysulfone or homogeneously modified polysulfone. The chemical modification was carried out by sulfonation and halomethylation. This additional change of the surface properties of the capillaries within the membrane offers the possibilities for various types of enzyme fixation, namely adsorption, charge interactions, or covalent bonding. By variation of the immobilization conditions the distribution of the enzyme could be adjusted over the membrane's cross section. At a distinct enzyme concentration in the loading solution a homogeneous enzyme distribution within the membrane could be verified. This was shown by diffusion experiments. Under ultrafiltration conditions using a solution that contains membrane-impermeable macromolecules as well as a membrane-permeable solute like saccharose the residence time within the membrane was increased due to gel formation atop the membrane yet the kinetic was no affected. The nonpermeable soluble starch was not reacted by the amyloglucosidase membrane, indicating that the skin layer was free of enzymes. (c) 1994 John Wiley & Sons, Inc.     

Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters

The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low-strength soluble wastewaters using ethanol as a model substrate was investigated in laboratory-scale reactors at 30oC. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to12 g COD/L . d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGBS reactor for the anaerobic treatment of low-strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate-biomass contact and adequate expansion of the substrate-biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (V(up)) was greater than 2.5 m/h. Under such conditions, an extremely low apparent K(s) value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at V(up) lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory-scale reactors. A. more important restriction of the EGSB reactor was the sludge washout occurring at V(up) higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L. d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold-up, the operation should be limited to an organic loading rate of 7 g COD/L d. and to a liquid up-flow velocity between 2.5 and 5.5 m/h (c) 1994 John Wiley & Sons, Inc.     

Predictions for oxygen supply control to enhance population stability of engineered production strains

The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of oxygen transfer in highly viscous non-newtonian fermentation broths

The influence of short draft tubes covered by perforated plates on gas-liquid mass transfer was examined in external-loop airlift bioreactors. The volumetric mass transfer coefficients in a model external-loop airlift bioreactor were measured with water and non-Newtonian media. It was found that introduction of draft tubes covered with perforated plates in the riser significantly improved the mass transfer rate, particularly in higher viscous non-Newtonian fermentation media. The enhancement of mass transfer rate might be due mainly to an increase in bubble coalescence and redispersion. (c) 1994 John Wiley & Sons, Inc.     

Localization of Drug-Metabolizing Enzyme Activities to Blood-Brain Interfaces and Circumventricular Organs

Abstract: The brain, with the exception of the choroid plexuses and Circumventricular organs, is partially protected from the invasion of blood-borne chemicals by the specific morphological properties of the cerebral micro-vessels, namely, the tight junctions of the blood-brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood-brain interfaces, where they form an "enzymatic barrier," is tested. By using microdissection techniques or a gradient-centrifugation isolation procedure, the activities of seven drug-metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three Circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three Circumventricular organs and cerebral microvessel than in the cortex. Very high membrane-bound epoxide hydrolase and UDP-glucuronosyltransferase activities (approaching those in liver) and somewhat high 7-benzoxyre-sorufin- O -dealkylase and NADPH-cytochrome P-450 reductase activities were determined in the choroid plexuses. The pia-arachnoid membranes, but not the dura matter, displayed drug-metabolizing enzyme activities, notably that of epoxide hydrolase: The drug-metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.     

A CYANOPHYTE CAPABLE OF FIXING NITROGEN UNDER HIGH LEVELS OF OXYGEN1

Nostoc cordubensis Prosperi (Cyanophyta, Nostocaceae) is characterized by its mucilaginous colonies. Much of this mucilage is produced by heterocysts. By controlling growth conditions, heterocysts with and without mucilage were obtained. Mucilaginous heterocysts retained nitrogen-fixing capability at high oxygen concentrations, whereas heterocysts lacking mucilage were unable to fix nitrogen at oxygen concentrations higher than 20%. Comparison of results obtained using tetrazolium salts as indicators of highly reduced zones showed similar results.     

Differences in the metabolic responses of root tips of wheat and rye to aluminium stress

The effects of aluminium (Al) ions on the metabolism of root apical meristems were examined in 4-day-old seedlings of two cereals which differed in their tolerance to Al: wheat cv. Grana (Al-sensitive) and rye cv. Dakowskie Nowe (Al tolerant). During a 24 h incubation period in nutrient solutions containing 0.15 mM and 1.0 mM of Al for wheat and rye, respectively, the activity of first two enzymes in the pentose phosphate pathway (G-6-PDH and 6-PGDH) decreased in the sensitive cultivar. In the tolerant cultivar activities of these enzymes increased initially, then decreased slightly, and were at control levels after 24 h. In the Al-sensitive wheat cultivar a 50% reduction in the activity of 6-phosphogluconate dehydrogenase was observed in the presence of Al. Changes in enzyme activity were accompanied by changes in levels of G-6-P- the initial substrate in the pentose phosphate pathway. When wheat was exposed for 16 h to a nutrient solution containing aluminium, a 90% reduction in G-6-P concentration was observed. In the Al-tolerant rye cultivar, an increase and subsequently a slight decrease in G-6-P concentration was detected, and after 16 h of Al-stress the concentration of this substrate was still higher than in control plants. This dramatic Al-induced decrease in G-6-P concentration in the Al-sensitive wheat cultivar was associated with a decrease in both the concentration of glucose in the root tips as well as the activity of hexokinase, an enzyme which is responsible for phosphorylation of glucose to G-6-P. However, in the Al-tolerant rye cultivar, the activity of this enzyme remained at the level of control plants during Al-treatment, and the decrease in the concentration of glucose occurred at a much slower rate than in wheat. These results suggest that aluminium ions change cellular metabolism of both wheat and rye root tips. In the Al-sensitive wheat cultivar, irreversible disturbances induced by low doses of Al in the nutrient solution appear very quickly, whereas in the Al-tolerant rye cultivar, cellular metabolism, even under severe stress conditions, is maintained for a long time at a level which allows for root elongation to continue.Abbreviations G-6-PDH glucose-6-phosphate dehydrogenase - 6-PGDH 6-phosphogluconate dehydrogenase - G-6-P glucose-6-phosphate - TEA triethanolamine     

Microbial indicators for sawdust and bark compost stability and humification processes

To what extent some microbial index ratios are suitable for use as early criteria for the level of compost stability during aerobic composting of coniferous sawdust and bark at mesophilic conditions was studied. Evolution of the specific respiration activity (CO₂-C/biomass C) and the ratios between some groups of microorganisms were followed as a function of composting time. The specific respiration activity was found to be an early and most reliable indicator of compost stability. The peroxidase and polyphenoloxidase enzyme activity during composting, as well as the composition of newly-formed humus substances were studied. The duration of composting increased the quality of newly-formed humus substances (C_h.a.:C_f.a ratio; Ca-complexed humic acid and resistance of organo-mineral complexes). The quality of humus substances could be used to assess compost stability. However, the results can be applied only under defined conditions.